Parallel confocal laser microscopy system based on vcsel technology

ABSTRACT

A parallel confocal laser microscopy system ( 2 ) includes a VCSEL vertical cavity laser array ( 23 ) for emitting light beams, optical elements ( 24 ) for focusing the light beams onto an object ( 25 ) to be observed. The invention is characterized in that a photodetector ( 22 ) is arranged behind each VCSEL laser such that the photodetector is capable of receiving a light beam backscattered from the object ( 25 ) via the VCSEL laser cavity, the cavity having an opening acting as filtering hole.

The present invention relates to a system and a method of parallelconfocal laser microscopy. It is used in particular, but notexclusively, in the field of medical imagery.

Generally, the principle of confocal microscopy is based on theillumination of a specimen by a point light source and by the detectionof the photons returning from this specimen through a filtering holeconjugate with an excitation plane, this allowing in particular a lightsection to be obtained.

The document “Parallel confocal laser microscope system using pixelarrays” by Makoto Naruse et al.; Proceedings of SPIE, Vol. 4092, pp.94-101 (on Internet:“http: //www.k2.u-tokyo.ac.jp/papers/optics/conf/naruse_confocal_SPIE00.pdf”) is known, in which the authordescribes a system 1 of parallel confocal microscopy in accordance withFIG. 1. An array 10 of lasers is shown of VCSEL type (“Vertical-cavitysurface-emitting laser”) emitting a light beam towards a specimen 13placed on a plate 14. This incident beam firstly passes through asemi-transparent mirror 11 then an optical system 12 to focus the beamonto the specimen 13. The mirror 11 allows the light beam back-scatteredby the specimen 13 to be deviated towards an array of photodetectors 16.In order to respect the notion of confocality, filtering holes 15 arearranged upstream of the photodetectors 16. A control unit 18 receivesthe signals generated by the photodetectors using a processing device 17so as to control the array of lasers 10, the plate 14 and the opticalsystem 12.

However, such a system is not optimized in terms of space requirement.

Moreover, document WO 0025165 (CNRS; Gorecki et al.) is known in whichan electronic component is described comprising a photodetector mountedon a VCSEL laser for the reception of a back-scattered beam originatingfrom a specimen. This component also comprises a tip for the emissionand the reception of the light beams. However, this document onlyrelates to microscopy in the near field without an optical system tofocus the light beams.

The purpose of the present invention is a miniature confocal microscopysystem.

Another purpose of the invention is to propose a microscopy systemallowing images to be acquired in real time.

The purpose of the invention is also to allow laser scanning for theacquisition of good quality images.

At least one of the aforementioned objectives is achieved with aparallel confocal laser microscopy system comprising in particular:

-   -   an array of vertical-cavity surface-emitting lasers (VCSEL) for        emitting light beams,    -   optical means for focusing the light beams onto an object to be        observed.

According to the invention, a photodetector is arranged on one face ofeach VCSEL laser such that this photodetector is capable of receiving alight beam originating from the object via the VCSEL laser cavity, thiscavity having an opening used as a filtering hole.

The invention is in particular remarkable because a virtual point lasersource is used the cavity opening of which serves as a filtering hole.The cavity opening of the VCSEL laser advantageously has a diameter of afew microns.

Preferably, the photodetector is arranged on a face opposite to thecavity opening of the VCSEL laser. Contrary to the system of FIG. 1 ofthe prior art, the laser source and the photodetector are aligned withthe optical axis, the laser beam axis. These two elements can beintegrated in the same device, which allows the space requirement of thesystem to be considerably reduced. The system can thus consist of aminiature head in the form of a housing. Therefore applications can beenvisaged such as endoscopy for which the miniature head is arranged atthe end of an endoscope. By way of example, the external diameter of theminiature head can be comprised between 2 and 10 mm, for a length ofbetween 10 and 30 mm.

As regards endoscopy, two implementation methods can be envisaged. Afirst method in which the miniature head is removable. In this case,this miniature head and its electric wiring (supply, control signals,useful signals etc.) can be inserted in the operating channel of anendoscope, the operating channel usually serving to pass through thetools which a practitioner needs to carry out measurements or takesamples. Therefore, the head is brought to the end of the endoscope soas to carry out, in particular, an optical biopsy. A second method inwhich the miniature head is fixed, completely integrated with the end ofan endoscope.

Generally, the system according to the invention can be used during thebackscattering applications.

Advantageously, the system can moreover comprise scanning means forcarrying out laser scanning so as to produce an image.

The array makes it possible, in particular, to work with a large amountof data at the same time, thus improving the quality of the imageobtained. In fact, it is possible to remain longer on each point and tointegrate for longer. The useful signal then contains enough informationto allow quality processing. Several points are acquired at the sametime whilst preserving the confocality, the latter being ensured by thevirtual point source assembly (spatial filtering) and optical system.The criterion of confocality can allow optical sections to be producedof the order of 1 to 3 microns. Thus, the choice of the VCSEL laser(useful cavity diameter and numerical aperture) and of the opticalsystem (magnification, numerical aperture) is in particular set by theconfocality.

Preferably, the system also comprises means for controlling the scanningmeans so as to carry out an acquisition of images in real time.

Depending on the array (parallel multi-point acquisition) and the typeof scanning used, the system makes it possible to go down to lowscanning frequencies such as for example 400 Hz, for which thecomponents are extremely reliable whilst allowing an acquisition ofimages in real time. By real time is meant an acquisition starting fromapproximately ten images per second. In order to reach such performances(approximately ten images per second), prior systems required scanningfrequencies over 4 kHz.

With respect to the system of the prior art (FIG. 1), the flux loss isreduced as the semi-transparent mirror disappears; the sensitivity ofdetection can be improved by increasing the integration time of the datasince the acquisition takes place over several points at the same time;and the image line scanning frequency can be adjusted in particulardownwards.

With the array according to the invention, the field of view can besufficiently large, i.e. having a surface of at least 150 microns by 150microns for example. The confocal character and a large enough field ofview represent a real advantage in the medical field, in particularwithin the context of helping with the early diagnosis of cancerouslesions.

Continuous scanning makes it possible to obtain an image in which eachpixel represented carries useful information originating from thespecimen.

The scanning frequencies and the number of laser sources can bedetermined so as to carry out an image acquisition in virtual real time.In certain fields such as the medical field, real time is a necessity inorder to compensate for the movement of the patient and thepractitioner.

Advantageously, the scanning means can comprise MEMS(“micro-electro-mechanical system”) micro-systems and/or piezoelectricpositioners, capable of moving the VCSEL laser array and/or the opticalmeans.

A person skilled in the art will easily understand that the opticalsystem can comprise one or more refractive and/or diffractive lenses.

According to the invention, the optical means, in particular the lenses,are capable of directing each light beam originating from the object tobe observed towards the cavity of a VCSEL laser, the cavity opening thencarrying out filtering.

Insofar as a photodetector is arranged to the rear of each VCSEL laser,the loss of light beams emitted to the rear of the VCSEL laser andcaptured by the photodetector are not negligible with respect to theuseful light beam originating from the object to be observed. Accordingto an advantageous characteristic of the invention, in order to onlydetect the useful light beam, means for modulating the light beamsleaving the array are arranged. These means can be an acousto-optical orelectro-optical modulator, or any other type of suitable modulationmeans. Thus, the light beams originating from the object to be observedare also modulated. Means of synchronous detection can then be arrangedto extract a useful signal from the electrical signal generated by eachphotodetector.

Advantageously, the optical means can comprise at least one moveablelens to allow an image acquisition at different depths of the object tobe observed. Therefore, three-dimensional images can be produced.Variable curvature lenses can also be used or the array can be movedaxially, i.e. along the z axis, to produce an in-depth scanning.

According to another aspect of the invention, a parallel confocal lasermicroscopy method is proposed in which a plurality of light beams isemitted from a VCSEL vertical cavity laser array, and these light beamsare focused onto an object to be observed using an optical system suchas lenses for example. According to the invention, a photodetector isarranged on a face of each VCSEL laser so as to receive a light beamoriginating from the object on this photodetector via the VCSEL lasercavity, and the opening of this cavity is used as a filtering hole forthe light beam originating from the object.

Preferably, the photodetector is arranged on the face opposite to theopening of the laser cavity.

Other advantages and characteristics of the invention will becomeapparent on examination of the detailed description of an embodimentwhich is in no way limitative, and of the attached drawings, in which:

FIG. 2 is a block diagram illustrating the operation of a microscopysystem according to the invention;

FIG. 3 is a diagram illustrating an example of the dimensioning of themain elements of a microscopy system according to the invention;

FIG. 4 a is a cross section of an electronic component comprising alaser of VCSEL type produced on a photodetector;

FIG. 4 b is a top view of the VCSEL laser of FIG. 4 a;

FIG. 4 c is a top view of a plurality of components of FIG. 4 a arrangedin an array;

FIG. 5 is a simplified diagram of the system according to the inventionin which the laser scanning is obtained by the movement of lenses usingMEMS systems; and

FIG. 6 is a simplified diagram of the system according to the inventionin which the laser scanning is obtained by movement of the array usingpiezoelectric positioners.

A miniature head according to the invention will now be described usingthe simplified and non-limitative diagrams of FIGS. 2 to 6.

The general principle of the system according to the invention isrepresented in FIG. 2. In contrast to the prior art as represented inFIG. 1, the system 2 according to the invention does not contain asemi-transparent mirror. In fact, in the system according to theinvention, the emitter i.e. the VCSEL laser, and the receiver i.e. thephotodetector, are aligned along the axis of the light beam. Eachphotodetector 22 is mounted on each VCSEL laser 23.

Each VCSEL laser 23 of the array emits a monochromatic and single-modelight which is focused by an optical system 24 onto the object to beobserved such as a specimen 25.

In particular VCSEL lasers are used with a wavelength comprised between630 nanometres and 1200 nanometres. The light beam back-scattered fromthe specimen 25 takes the same path as the incident beam via the opticalsystem 24, then returns into the VCSEL laser 23 passing through it untilreaching the photodetector 22.

The electrical signal generated by the photodetector 22 is processed bya processing system 21 comprising, in particular, means of amplificationand digitizing. The digital signal 29 is then transmitted to a controlunit 26. The array, composed of the elements 22 and 23, and the opticalsystem 24 are capable of being controlled by the control unit 26 usingcontrol signals 28 and 27 respectively. The control signals 28 canconsist of commands for moving the array in two directions x and y so asto acquire two-dimensional images (as seen in FIG. 6), signalscontrolling the light intensity of the VCSEL lasers, signals controllingthe photodetectors and signals controlling the processing means. Thecontrol signals 27 are capable of managing the movement of the opticalsystem as seen in FIG. 5.

The array and the optical system can be integrated into a miniature head20 arranged at the end of an endoscope.

In FIG. 3 an example of the dimensioning of a system according to theinvention is represented. In this example, the optical system containstwo diffractive lenses. The dimensioning parameters are the following:

Wavelength: between 680 and 880 nm;

Source field: 2ΔX=400-600 μm

Diameter of the VCSEL cavity opening: Φ_(cavity) =2-4 μm

VCSEL cavity numerical aperture: sin(α)=0.25 (in air)

Focal length first lens: f1=3 mm

Diameter first lens: Φ_(total)=2 mm

Focal length second lens: f2=1.17 mm

Diameter second lens: Φ2=1.6 mm

Optical system magnification: G=3

Imaged field: 2ΔX_(object)=160 μm-240 μm

Object numerical aperture=n sin(α₂)=0.75 (in water, n=1.33)

The diameter of each spot focused in the specimen is limited by thediffraction over all the field imaged.

With a system as represented in FIG. 2, in order to produce an image,laser scanning is carried out either by moving the lenses of the opticalsystem 24 using MEMS systems as will be seen further on in FIG. 5, or bymoving the array with piezoelectric positioners as will be seen in FIG.6. The scanning frequencies are chosen as a function of the number ofpoint sources (VCSEL laser) used simultaneously in the array. Forexample, for a 10 by 10 array, frequencies of 10 hertz ((frame) and 400hertz (line) are used. These frequencies make it possible to obtain areal-time two-dimensional scanning.

The signal originating from the specimen is focused at the VCSEL laserinput by taking the same optical path as the incident signal. Thespatial filtering necessary to the confocality is carried out at theinput/output of the VCSEL laser as the cavity opening of this laser isof the order of a few microns. The confocality is dependent on thenumerical aperture and the magnification of the optical system as wellas on the numerical aperture of the lasers. The signal thus filtered isthen detected by the photodetector which is placed behind the lasercavity.

The amplification factor of the VCSEL laser cavity is approximately 10⁶.

Two detection modes can be envisaged such as:

-   -   Continuous mode: the VCSEL laser emits continuously. Part of        this emitted light is detected by the photodetector as the Bragg        mirror in the cavity on the detector side has transmission of        the order of 1%. The background signal detected by the        photodetector and originating from the cavity is of the order of        10⁻². On the other hand, the back-scattered signal originating        from the specimen being of the order of 10⁻⁵ to 10⁻⁶, this        signal is amplified by the cavity until it reaches a value        comprised between 1 and 10 in the cavity. Passing through the        Bragg mirror causes its value to pass between 10⁻² and 10⁻¹. The        useful signal generated by the photodetector is therefore at        least of the order of the background signal.    -   Synchronous mode: the output signal of the VCSEL laser is        modulated by an acousto-optical modulator (not shown) placed in        the optical system 24. The useful signal is itself therefore        also modulated at the same frequency. It is then sufficient to        use detection synchronous with the modulation signal in order to        extract the useful signal and reject the background signal.

FIG. 4 a shows in slightly more detail an electronic component accordingto the invention in which, starting with the same substrate, aphotodetector and a VCSEL laser are produced by epitaxial growth. Thephotodetector is arranged on the rear face of the laser opposite to theemission face of the laser. FIG. 4 b is a front view of the electroniccomponent of FIG. 4 a. It shows in particular the cavity opening of theVCSEL laser through which the light beam is emitted. For information,the diameter of this opening can be comprised between 2 and 8micrometres whereas the electronic component can have an overall lengthof 50 microns. FIG. 4 c is a front view of several electronic componentsof FIG. 4 a arranged in an array. With the dimensions of FIG. 4 b and byarranging the components in a 10 by 10 array, an array is obtained theside of which is equal to 500 microns, making it possible to obtain alarge enough field of view.

FIG. 5 shows a miniature head according to the invention in which thelaser scanning is obtained by movement of two lenses. The miniature headin FIG. 5 comprises a housing 50, at the base of which a VCSELlaser/photodetector array 51 is arranged. The lasers in the array emitalong parallel axes towards the inside of the housing 50. The lightbeams emitted pass through three lenses 52, 53 and 54 so as to befocused on an object (not shown) outside the housing on the other sideof an exit window 55 arranged on the base opposite to the basecontaining the array 51. The light beams all converge in an image fieldplane arranged in the object to be observed (not shown).

The convergence lens 54 is fixed integral with the housing 50, whereasthe two lenses 52 and 54 are mobile, being fixed on MEMS micro-systems56 and 57. The MEMS 56 allows the lens 52 to be moved in an X directionin a plane perpendicular to the laser emission axis. The MEMS 57 allowsthe lens 53 to be moved in a Y direction perpendicular to the laseremission axis and the X axis. These movements make it possible to carryout laser scanning in an X Y plane. Then, by processing the signalupstream of the photodetectors, the image field can be reconstructed.The scanning amplitude of MEMS micro-systems is determined so as toachieve at least 150 by 150 microns of imaged fields for example. Thedata processing can consist of conventional algorithms.

According to a variant or in a complementary fashion, the laser scanningcan be operated by movement of the array. In order to do this, theminiature head is a housing 60, at the base of which an array 61 isarranged. Piezoelectric positioners are inserted between the lateralfaces of the array and the housing 60. These piezoelectric positionersare arranged in twos on parallel sides. The positioners 62 allowmovement along the X axis, and the positioners 63 allow movement of thearray along the Y axis. In this case, the lenses 64 and 65 allowing thelight beams to be focused can be fixed.

Then only two lenses are used instead of three as previously. Theamplitude of movement of the piezoelectric positioners allows each VCSELlaser to be covered. By way of example, with the dimensions in FIGS. 4 ato 4 c, this amplitude is approximately 50 microns.

The laser scanning operations as represented in FIGS. 5 and 6 allow atwo-dimensional image of the imaged field to be obtained. Light beamscanning can then be introduced in an axial direction in order to choosethe depth of visualization in the object to be observed. Scanning in theZ direction perpendicular to the X and Y directions allowsthree-dimensional reconstructions of the object observed. In order to dothis, different two-dimensional acquisitions are carried out atdifferent depths and a volume is reconstructed by data processing.

More precisely, again with reference to FIGS. 5 and 6, in-depth scanningis carried out by making the lens 54 in FIG. 5, the lens 64 or the lens65 in FIG. 6 mobile. This new mobile lens makes it possible to focus allof the scanned field laterally (two-dimensional image) at differentdepths in the object observed. The movement of this lens can be obtainedusing piezoelectric elements or MEMS micro-systems. Scanning in the Zdirection can be carried out according to two modes: either by changingthe depth of visualization frame by frame, the acquisitions beingcarried out at given depths, or by making in-depth “films”, i.e. theacquisition is carried out automatically on different successive planesbefore three-dimensional reconstruction.

Of course, the invention is not limited to the examples which have justbeen described and numerous adjustments can be made to these exampleswithout exceeding the framework of the invention. In fact, it ispossible to envisage increasing the complexity of the optical means inorder to optimize the performance of the system, in particular in orderto avoid any aberration faults, as a function of the application of thesystem.

1. Parallel confocal laser microscopy system comprising: an array ofvertical-cavity lasers (VCSEL) for emitting light beams, and opticalmeans for focusing the light beams onto an object to be observed.characterized in that a photodetector is arranged on one face of eachVCSEL laser such that this photodetector is capable of receiving a lightbeam originating from said object via the VCSEL laser cavity, thiscavity having an opening used as a filtering hole.
 2. System accordingto claim 1, characterized in that the photodetector is arranged on aface opposite to the cavity opening of the VCSEL laser.
 3. Systemaccording to claim 1, characterized in that it moreover comprisesscanning means for carrying out laser scanning so as to produce animage.
 4. System according to claim 1, characterized in that it moreovercomprises means for controlling the scanning means so as to carry out anacquisition of images in real time.
 5. System according to claim 3characterized in that the scanning means comprise MEMS micro-systems. 6.System according to claim 3, characterized in that the scanning meanscomprise piezoelectric positioners.
 7. System according to claim 3,characterized in that the scanning means are capable of moving the VCSELlaser array.
 8. System according to claim 3, characterized in that themeans of scanning are capable of moving optical means.
 9. Systemaccording to claim 1, characterized in that the optical means arecapable of directing each light beam originating from the object to beobserved towards the cavity of a VCSEL laser.
 10. System according toclaim 1, characterized in that it moreover comprises modulation meansfor modulating the light beams emitted from the array.
 11. Systemaccording to claim 10 in which the light beams originating from theobject to be observed are modulated, characterized in that it comprisessynchronous detection means for extracting a useful signal from theelectrical signal generated by each photodetector.
 12. System accordingto claim 1, characterized in that the optical means comprise at leastone mobile lens for allowing image acquisition at different depths ofthe object to be observed.
 13. System according to claim 1,characterized in that the optical means comprise at least one variablecurvature lens for allowing image acquisition at different depths of theobject to be observed.
 14. System according to claim 1, characterized inthat it comprises means for axially moving the array so as to carry outimage acquisition at different depths of the object to be observed. 15.System according to claim 1, characterized in that it consists of aminiature head in the form of a housing.
 16. Application of the systemaccording to claim 15 in which the miniature head is arranged at the endof an endoscope.
 17. Method of parallel confocal laser microscopy inwhich a plurality of light beams are emitted from an array of VCSELvertical cavity lasers, these light beams are focused on an object to beobserved; characterized in that a photodetector is arranged on a face ofeach VCSEL laser so as to receive a light beam originating from theobject on this photodetector via the VCSEL laser cavity, and in that theopening of this cavity is used as a filtering hole for the light beamoriginating from the object.
 18. Method according to claim 17,characterized in that laser scanning is carried out so as to produce animage.
 19. Method according to claim 17, characterized in that laserscanning is carried out so as to acquire images in real time.
 20. Methodaccording to claim 18 characterized in that the laser scanning iscarried out by moving optical means used to focus the light beams. 21.Method according to claim 18, characterized in that the laser scanningis carried out by moving the array.
 22. Method according to claim 18,characterized in that MEMS-type micro-systems are used for carrying outthe laser scanning.
 23. Method according to claim 18, characterized inthat piezoelectric positioners are used for carrying out the laserscanning.
 24. Method according to claim 17, characterized in that thelight beams emitted from the array are modulated and synchronousdetection is carried out at the level of the photodetector.